The generation of neural connectivity critically depends upon temporally and spatially coordinated regulation of neuronal process guidance. The central goal of this proposal is to understand the functions and mechanisms employed by specific neuronal semaphorin guidance cues to regulate axonal and dendritic targeting. A range of cues and their receptors play key roles in attracting and repelling neuronal processes as they extend toward their final targets, where guidance cues also play important roles in target recognition and synaptogenesis. During the previous funding period, we identified unique roles for semaphorin cues and their receptors in guiding neuronal processes in both Drosophila and in the mouse, and we also investigated signaling components that serve to facilitate these guidance events. Our cross-phylogenetic approach provides unique insight into select guidance cue-mediated neuronal targeting, and it uses strong complementary experimental strategies. In Drosophila, we have the ability to employ powerful molecular and genetic approaches to address complex aspects of guidance cue function and receptor signaling in vivo. In the mouse, we have the opportunity to employ one of the best characterized laminar structures in the nervous system, the vertebrate retina, to investigate the molecular mechanisms underlying the assembly of complex neuronal connectivity, utilizing powerful genetic and anatomical strategies. Our studies during the previous funding period raise several issues we propose investigating in this renewal application of our long-standing work on semaphorin- mediated neuronal guidance. Our results show that closely related Drosophila secreted semaphorins function through the same receptor to mediate short-range attraction or longer-range repulsion, and we have in place both in vivo and in vitro experimental paridigms that will allow us to begin to dissect critical ligand and receptor signaling interactions that lead to divergent guidance cue responses (Aim I). Our work on mouse transmembrane semaphorin regulation of retinal lamination raises intriguing issues regarding novel mechanisms by which distinct classes of retinal ganglion cells (RGCs) and amacrine cells establish their exquisite connectivity in the IPL, and also how select RGC axonal projections employ transmembrane semaphorins and their plexin receptors to regulate targeting to appropriate retinorecipient CNS targets (Aim II). These studies will contribute to our understanding of circuit assembly and function.